The aims of this study were to 1) directly assess active muscle stiffness according to actual length changes in muscle fibers (fascicles) during short range stretching; and 2) compare actual measured active muscle and tendon stiffness using ultrasonography with the stiffness of active (i.e., muscle) and passive (i.e., tendon) parts in series elastic component of plantar flexors using the alpha method. Twenty-four healthy men volunteered for this study. Active muscle stiffness in the medial gastrocnemius muscle was calculated according to changes in estimated muscle force and fascicle length during fast stretching after submaximal isometric contractions [10, 30, 50, 70, and 90% maximal voluntary contractions (MVC)]. Using the variables measured during this fast stretch experiment, the stiffness of active (i.e., muscle) and passive (i.e., tendon) parts in plantar flexors was assessed using alpha method. Tendon stiffness was determined during isometric plantar flexion by ultrasonography. Active muscle stiffness increased with the exerted torque levels. At 30, 50, 70, and 90% MVC, there were no significant correlations between muscle stiffness using ultrasonography and stiffness of active part (i.e., muscle) by alpha method, although this relationship at 10% MVC was significant (r = 0.552, P = 0.005). In addition, no correlation was noted in tendon stiffness between the two different methods (r = 0.226, P = 0.209). The present study demonstrated that ultrasonography could quantified active muscle stiffness in vivo. Furthermore, active muscle stiffness and tendon stiffness using ultrasonography were not related to active (i.e., muscle) or passive (i.e., tendon) stiffness in series elastic component of plantar flexors by alpha method.